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Product FAQ

How does the optical density (OD) of a culture relate to the number of cells for Saccharomyces cerevisiae?

Answer

OD600 of 0.1 = approximately 3 x 10e6 cells/mL

Answer Id: E9548

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Can old premixed lithium acetate buffers be used for preparing and transforming Saccharomyces cerevisiae?

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Stock buffers of TE, lithium acetate, and PEG can be stored. However, the combined solution used to prepare the cells for transformation must be made fresh every time. There is a loss in transformation efficiency if the solutions are not freshly prepared.

Answer Id: E9549

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For galactose induction of expression in Saccharomyces cerevisiae, is it possible to include additional carbon sources in the media that will increase yeast growth without repressing expression from the GAL promoter?

Answer

Some researchers choose to grow yeast in medium containing 2% galactose as the sole carbon source during induction. However, yeast typically grow more quickly in induction medium containing 2% galactose plus 2% raffinose. Raffinose is a good carbon source for yeast, and unlike glucose, does not repress transcription from the GAL promoter. Raffinose is a trisaccharide of galactose, glucose and fructose linked in that order. Most yeasts can cleave the glucose-fructose bond, but not the galactose-glucose bond. Fructose is then used as a carbon source.

Answer Id: E3734

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Which S. cerevisiae yeast strain do your kits contain?

Answer

We offer the INVSc1 yeast strain. It is a diploid strain for expression purposes only. It does not sporulate well and is therefore not suited for yeast genetic studies. The genotype and phenotype of the INVSc1 strain are as follows:

Genotype: MATa his3?1 leu2 trp1-289 ura3-52/MATalpha his3?1 leu2 trp1-289 ura3-52
Phenotype: His-, Leu-, Trp-, Ura-
Note that INVSc1 is auxotrophic for histidine, leucine, tryptophan, and uracil. The strain will not grow in SC minimal medium that is deficient in histidine, leucine, tryptophan, and uracil.

Answer Id: E9550

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What choices do you offer for protein expression in a yeast host system, and what are their features?

Answer

We offer the original Pichia pastoris expression systems, PichiaPink™ expression system, and Saccharomyces cerevisiae yeast expression system for expression of recombinant proteins. Both P. pastoris and S. cerevisiae have been genetically well-characterized and are known to perform many posttranslational modifications.

The P. pastoris expression system combines the benefits of expression in E. coli (high-level expression, easy scale-up, and inexpensive growth) and the advantages of expression in a eukaryotic system (protein processing, folding, and posttranslational modifications), thus allowing high-level production of functionally active recombinant protein. As a yeast, Pichia pastoris shares the advantages of molecular and genetic manipulations with Saccharomyces cerevisiae, and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia pastoris very useful as a protein expression system. The Pichia expression vectors contain either the powerful alcohol oxidase (AOX1) promoter for high-level, tightly controlled expression, or the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for high-level, constitutive expression. Both inducible and constitutive expression constructs integrate into the P. pastoris genome, creating a stable host that generates extremely high protein expression levels, particularly when used in a fermentor. The Pichia pastoris expression systems we offer include:

- PichiaPink™ Yeast Expression System: Newer Pichia pastoris expression system that contains both low- and high-copy plasmid backbones, 8 secretion signal sequences, and 4 yeast strains to help optimize for the highest yield possible of the recombinant protein. All PichiaPink™ vectors contain the AOX1 promoter for high-level, inducible expression and the ADE2 marker for selecting transformants using ADE2 complementation (i.e., by complementation of adenine auxotrophy) rather than antibiotic selection. However, they express the ADE2 gene product from promoters of different lengths, which dictate the copy number of the integrated plasmids. The pPink-LC vector has an 82 bp promoter for the ADE2marker and offers low-copy expression, and the pPink-HC vector has a 13 bp promoter for the ADE2marker and offers high-copy expression. The system also includes the pPinkalpha-HC vector (containing S. cerevisiae alpha-mating factor pre-sequence) for high copy number secreted expression, and provides eight secretion signal sequences for optimization of secreted expression.
- EasySelect™ Pichia Expression Kit: One of the original Pichia expression kits that contains the pPICZ and pPICZalpha vectors, for intracellular and secreted expression, respectively, of the gene of interest. These vectors contain the AOX1 promoter for high-level, inducible expression and the Zeocin™ antibiotic resistance marker for direct selection of multi-copy integrants. They facilitate simple subcloning, simple purification, and rapid detection of expressed proteins.
- Original Pichia Expression Kit: The kit includes the pPIC9, pPIC3.5, pHIL-D2, and pHIL-S1 vectors, each of which carries the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. pPIC9 carries the S. cerevisiae alpha-factor secretion signal while pHIL-S1 carries the Pichia pastoris alkaline phosphatase signal sequence (PHO) to direct transport of the protein to the medium. pHIL-D2 and pPIC3.5 are designed for intracellular expression.
- Multi-Copy Pichia Expression Kit: This kit is designed to maximize expression and contains the pPIC3.5K, pPIC9K, and pAO815 vectors, which allow production and selection of Pichia strains that contain more than one copy of the gene of interest. They allow isolation and generation of multicopy inserts by in vivo methods (pPIC3.5K and pPIC9K) or in vitro methods (pAO815). All of these vectors contain the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K and pAO815 remain intracellular. The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance marker that confers resistance to Geneticin™ Reagent in Pichia. Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin™ Reagent. Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to Geneticin™ Reagent. The ability to grow in high concentrations of Geneticin™ indicates that multiple copies of the kanamycin resistance gene and the gene of interest are integrated into the genome.
- For expression in S. cerevisiae, we offer the pYES™ Vector Collection. Each pYES™ vector carries the promoter and enhancer sequences from the GAL1 gene for inducible expression. The GAL1 promoter is one of the most widely used yeast promoters because of its strong transcriptional activity upon induction with galactose. pYES™ vectors also carry the 2m origin and are episomally maintained in high copy numbers (10-40 copies per cell).

Answer Id: E9478

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Does pYES2 have a CEN sequence?

Answer

pYES2 does not have a CEN sequence. pYES2 has the 2μ ori which maintains a copy number of 30 to 50 copies per cell. The 2μ ori sequence is derived from the endogenous 2μ circle plasmid (it's the replication origin for 2μ circle).

'CEN' stands for 'CENtromere' sequence. This origin of replication keeps the copy number of that vector down to one or two per cell in yeast. CEN sequences are actual chromosome centromere sequences. CEN ori will keep copy number to 1 or 2 per cell (depending on whether the cell is haploid or diploid). This sequence allows the cell to recognize this plasmid as a chromosome; regulation of chromosome number is extremely rigid. Only functional CEN sequences from S. cerevisiae have been isolated and used on plasmids. CEN sequences are only a couple of hundred base pairs in size. Apparently functional centromere sequences from other eukaryotes, including Sc. pombe and Pichia, are too large to be isolated and utilized on a plasmid.

Answer Id: E3775

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Will S. cerevisiae grow differently using galactose instead of glucose as a carbon source?

Answer

S. cerevisiae can grow using either or both mechanisms of carbon metabolism. The balance between the two is different for glucose vs. galactose as a carbon source. Under ideal conditions, S. cerevisiae grows slower on galactose than on glucose, because production of glucose-6-P from galactose is rate limiting. (gal -> gal-1-P -> glu-1-P -> glu-6-P). Under non-ideal conditions (low oxygen, as in the center of a colony or a culture without really good oxygen feed), it becomes even worse because cells grown on galactose are using more respiration than fermentation relative to cells grown on glucose. Low oxygen makes fermentation more necessary, which cells growing on galactose are not good at.

Answer Id: E4309

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What is the doubling time of pYES2 transformed S. cerevisiae strain on minimal yeast growth media when either glucose or galactose is used as the carbon source?

Answer

The doubling time of a pYES2 transformed S. cerevisiae strain grown on minimal media with glucose is approximately 2 hours. The doubling time on media with galactose is approximately 4 hours.

Answer Id: E3926

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What are the different kinds of media used for culturing Pichia pastoris and S. cerevisiae?

Answer

Following are the rich and minimal media used for culturing Pichia pastoris and S. cerevisiae:

Rich Media:
S. cerevisiae and Pichia pastoris
YPD (YEPD): yeast extract, peptone, and dextrose
YPDS: yeast extract, peptone, dextrose, and sorbitol

Pichia pastoris only
BMGY: buffered glycerol-complex medium
BMMY: buffered methanol-complex medium

Minimal Media (also known as drop-out media):
S. cerevisiae
SC (SD): Synthetic complete (YNB, dextrose (or raffinose or galactose), and amino acids)

Pichia pastoris
MGY: minimal glycerol medium
MD: minimal dextrose
MM: minimal methanol
BMGH: buffered minimal glycerol
BMMH: buffered minimal methanol

Answer Id: E9556

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Product FAQ

What could explain bands of unexpected molecular weight appearing in galactose induced pYES transformants?

Answer

Although there are no other genes on the plasmid that are induced, there certainly are a number of proteins in the cell that are turned on by galactose and any of those may be apparent. As with any experiment, one should always run the plasmid without insert, induced with galactose as a negative control. The additional bands could be due to post-translational modifications, such as glycosylation, that would increase the apparent molecular weight. Since the size of the sugar chains can be variable, glycosylated proteins often appear as less well defined bands compared to non-glycosylated proteins.

Answer Id: E3776

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Product FAQ

For galactose induction of expression in S. cerevisiae, can I include additional carbon sources in the media to increase yeast growth without repressing expression from the GAL promoter?

Answer

Some researchers choose to grow yeast in medium containing 2% galactose as the sole carbon source during induction. However, yeast typically grow more quickly in induction medium containing 2% galactose plus 2% raffinose. Raffinose is a good carbon source for yeast, and unlike glucose, does not repress transcription from the GAL promoter. Raffinose is a trisaccharide of galactose, glucose, and fructose linked in that order. Most yeast can cleave the glucose-fructose bond, but not the galactose-glucose bond. Fructose is then used as a carbon source.

Answer Id: E9552

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What range of efficiency of transformation I should expect when preparing and electroporating Saccharomyces cerevisiae?

Answer

The efficiency is very strain-dependent, but 1000 to 100,000 transformants per μg DNA is the range.

Answer Id: E9547

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Product FAQ

Should D-raffinose be used as carbon source for yeast prior to galactose induction? Does L-raffinose work?

Answer

As with other sugars (e.g. glucose), D-raffinose is the biologically active carbon source for yeast. Pure L-raffinose will not work.

Answer Id: E3779

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Why would I pick a yeast expression system for expression of my protein, as opposed to expression systems in other hosts?

Answer

Yeast is a single-celled, eukaryotic organism that can grow quickly in defined media (doubling times are typically 2.5 hr in glucose-containing media) and is easier and less expensive to use for recombinant protein production than insect or mammalian cells (see table below). These positive attributes make yeast suitable for use in formats ranging from multi-well plates, shake flasks, and continuously stirred tank bioreactors to pilot plant and industrial-scale reactors.

The most commonly employed species in the laboratory are Saccharomyces cerevisiae (also known as Baker’s or Brewer’s yeast) and some methylotrophic yeasts of the Pichia genus. Both S. cerevisiae and P. pastoris have been genetically characterized and shown to perform the posttranslational disulphide bond formation and glycosylation that is crucial for the proper functioning of some recombinant proteins. However, it is important to note that yeast glycosylation does differ from that in mammalian cells: in S. cerevisiae, O-linked oligosaccharides contain only mannose moieties, whereas higher eukaryotic proteins have sialylated O-linked chains. Furthermore S. cerevisiae is known to hyperglycosylate N-linked sites, which can result in altered protein binding, activity, and potentially yield an altered immunogenic response in therapeutic applications. In P. pastoris, oligosaccharides are of much shorter chain length and a strain has been reported that can produce complex, terminally sialylated or “humanized” glycoproteins.

Answer Id: E9477

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Product FAQ

How do I store yeast long term? Can I freeze them at -80 degrees C like bacteria? What is the shelf life of frozen stocks?

Answer

We recommend storing yeast frozen at -80 degrees C in 15% glycerol. Glycerol stocks are good indefinitely (unless there are numerous freeze-thaws). When making a glycerol stock, we recommend using an overnight culture and concentrating it 2-4 fold. Spin down cells and suspend in 25-50% of the original volume with glycerol/medium. It is better to store frozen cells in fresh medium plus glycerol, rather than simply adding glycerol into the overnight culture.

Answer Id: E9480

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